JP5700763B2 - Antiallergic agent - Google Patents

Description

  The present invention relates to an antiallergic agent mainly composed of acidic polysaccharides.

Currently, 20-30% of people in Japan are considered to have some allergic symptoms, and the increase in familiar hay fever and atopic dermatitis is one example. This increase in allergic symptoms is a major social problem.
In general, allergy refers to immediacy that is classified as type I in allergic reactions that are classified into four in terms of immunology. Type I allergy is caused by contact with allergens contained in pollen, ticks, eggs, milk, etc., to induce an immune response in humans and the like, and to produce IgE, which is an allergy-causing antibody. The produced IgE is transported throughout the body and then binds via Fc receptors expressed on mast cells and basophils. The allergen taken into the body again crosslinks with this IgE, thereby promoting the release of histamine stored in mast cells or basophils and the production of leukotrienes, causing immediate allergic symptoms. Will be.

To date, various drugs and food materials have been developed for the purpose of alleviating, suppressing, and treating type I allergic symptoms, and pectin is one of them.
The antiallergic activity of pectin has been suggested or confirmed by a test using animal cells (in vitro) or a test taken in mice, humans, etc. (in vivo).
In Non-Patent Document 1, it has been confirmed in experiments using animal cells that pectin suppresses the release of histamine, which is considered as an index of antiallergic activity. Patent Literature 1 discloses that polygalacturonic acid inhibits a specific antigen-antibody reaction with a cedar pollen antigen and suppresses histamine release by a test using venous blood of a hay fever patient.
Furthermore, in Patent Document 2, as a result of ingesting 22.2 g / day of apple-derived pectin (9.99 g / day as a pectin equivalent amount) for 3 weeks to a human (adult), the histamine concentration in plasma decreases, It has been confirmed that it has a therapeutic or preventive effect on allergic diseases.
In Non-Patent Document 2, it has been confirmed that when a diet containing 5% pectin is administered to a rat for 2 weeks, IgE in serum decreases and IFN-γ of lymphocytes increases significantly. In this test, pectin was administered at a rate of about 7.2 g / kg body weight (calculated value).
And in patent document 3, while applying an antigen to an ear | edge with respect to a mouse | mouth, the amount equivalent to 100 mg / kg of pectin of 35% of esterification is suspended in water, and it is orally administered by a sonde for a total of 9 times over 4 weeks. As a result, it was confirmed that the production of IgE as an antibody was suppressed and the Th1 / Th2 balance was improved.
Thus, it has been confirmed that pectin has an effect of suppressing histamine release from mast cells, which is the direct cause of the development of allergy, and an immune improvement effect of suppressing the production of antibodies involved in the development of allergy.
However, in the disclosed administration test to animals and humans, it takes two weeks or more until the antiallergic effect is exhibited, and the effect is expected if it is not ingested more than two weeks before the onset of allergy. Therefore, there is a demand for the development of a substance that shows an effect in a shorter time.

The present inventors have found that when an acid or saline-soluble polymer substance obtained from soybean is orally administered to mice, it exhibits antiallergic activity (Japanese Patent Application No. 2009-135724). Although it is considered that the main component of the polymer substance obtained in this production method is a polysaccharide, specific components have not been specified.
In addition, high molecular polysaccharides contained in soy sauce have antiallergic and immunomodulating effects (Patent Documents 4 and 5), and this substance is an acidic polysaccharide containing galacturonic acid, and has a molecular weight of 50,000 or less. It is disclosed that it exhibits strong antiallergic activity (Non-patent Documents 3 and 4), but no further components are specified, and it has antiallergic activity obtained from fermented soybeans such as soybeans and soy sauce. Among substances, it has been desired to identify substances that are particularly involved in antiallergic activity and to obtain substances having stronger antiallergic activity.

Japanese Patent Application Laid-Open No. 07-330617 JP 2004-107295 A JP 2001-233777 A JP 2003-327540 A JP 2007-084486 A

Y. Sawabe et al., Biochim. Biophys. Acta. 1137, 274-278, 1992. B. O. Lim et al. Nutr. 663-667, 1997 Abstracts of Annual Meeting of the Japan Society for Biotechnology 2007 Annual Meeting, p172 Agricultural Chemistry Society of Japan Kansai Chapter 5 (2005)

  An object of the present invention is to provide a highly active antiallergic agent that exerts an effect in a short period of time compared to conventional pectin-related antiallergic agents.

As a result of intensive studies to solve the above-mentioned problems, the present inventors further fractionated a substance having antiallergic activity obtained from soybean fermented products such as soybean or soy sauce with an anion exchange resin, It has been found that an acidic fraction having high antiallergic activity can be obtained. In addition, by analyzing this acidic fraction, 1) containing an acidic polysaccharide as a main component, 2) soluble at pH 2, and 3) containing galacturonic acid and xylose as constituents, xylose / galacturonic acid The present inventors have found that an antiallergic agent characterized by having a weight ratio of 0.2 or more can be obtained, thereby completing the present invention.
It has not been known that pectin having a high xylose / galacturonic acid ratio and soluble in acid as in the present invention has strong antiallergic activity. Also, it is a novel anti-allergic agent with new characteristics because it has a different constituent sugar composition from commercially available soybean polysaccharides, and has a distinctly different acid solubility and constituent sugar composition from pectin obtained from citrus, etc. confirmed.

(1) An antiallergic agent having the following features 1) to 3).
1) Mainly composed of acidic polysaccharide 2) Soluble at pH 2 3) Containing galacturonic acid and xylose as constituent components, and xylose / galacturonic acid weight ratio of 0.2 or more (2) galacturonic acid The antiallergic agent according to (1) above, containing 10% or more.
(3) The antiallergic agent according to (1) or (2), which is obtained using soybean as a raw material.
(4) The antiallergic agent according to any one of (1) to (3) obtained by extracting soybean or soybean seed coat with acidic water.
(5) The antiallergic agent according to any one of (1) to (3), which is obtained by extraction from a fermented soybean product.

  The anti-allergic agent of the present invention exhibits strong anti-allergic activity when administered orally for a short period such as 3 days, unlike the anti-allergic agent obtained from conventional fermented soybeans such as pectin, soybean and soy sauce. The present invention includes 1) an acidic polysaccharide as a main component, 2) is soluble at pH 2, and 3) contains galacturonic acid and xylose as constituents, and the weight ratio of xylose / galacturonic acid is 0.2 or more. The provision of a novel antiallergic agent characterized by that makes it possible to provide a more useful drug for allergic symptoms.

The “antiallergic agent” of the present invention refers to an agent useful for preventing the occurrence of allergic symptoms, alleviating symptoms, treating them, and the like.
Any one having the following features 1) to 3) is included in the “antiallergic agent” of the present invention.
1) Mainly composed of acidic polysaccharides 2) Soluble at pH 2 3) Containing galacturonic acid and xylose as constituent components, and the weight ratio of xylose / galacturonic acid is 0.2 or more

Here, the “acidic polysaccharide” is a high-molecular acidic polysaccharide, and any one containing galacturonic acid at a high ratio of 10% or more, preferably 15% or more, more preferably 20% or more. Applicable examples include pectin and the like.
Further, “soluble at pH 2” may be soluble in any aqueous solution as long as it is a pH 2 solution, and examples thereof include solutions such as an aqueous citric acid solution at pH 2 and an HCl solution.
Both galacturonic acid and xylose are contained as constituents, and the weight ratio of xylose / galacturonic acid should be 0.2 or more, particularly 0.2 or more and less than about 1.0. preferable.

  The antiallergic agent of the present invention is preferably obtained from soybean as a raw material, and can be obtained by extracting soybean itself, soybean seed coat removed or soybean seed coat removed with acidic water. Moreover, the soybeans heated by steaming etc. can also be used, and it can also extract and obtain from soybean fermented products, such as soy sauce, miso, and natto.

  The antiallergic agent of the present invention may be produced using any conventionally known method, equipment, etc., as long as it can produce the “antiallergic agent” of the present invention. For example, when the acidic fraction is obtained, any resin can be used for the fraction as long as it is an anion exchange resin.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

Manufacture of antiallergic agents (1)
9 parts of 3% aqueous citric acid solution (about pH 2) was added to 1 part of the ground soybean seed coat, heated at 90 ° C. for 5 hours, and solid-liquid separation yielded about 6 parts of clear liquid. This was dialyzed with a dialysis membrane having a molecular weight cut off of 6000 for 20 hours in running water, added to the dialysis internal solution with 95% ethanol twice as much, stirred and allowed to stand, and the generated precipitate was collected by centrifugation and dried ( In Test Example 1, it was designated as Sample 1.) A part of this was fractionated with an anion exchange resin, and the acidic fraction adsorbed on the anion exchange resin was used as the antiallergic agent of the present invention.
Fractionation with this anion exchange resin was performed by HPLC fractionation using a 21.5 × 150 mm column (TOSOH) packed with TSKgel DEAE-5PW. As the eluent, a linear gradient of 50 mM acetate buffer (pH 5.0) and 50 mM acetate buffer (pH 5) to which 1M sodium acetate was added was used (flow rate and concentration gradient are described). Fractionated into a non-adsorbed part and an adsorbed part, dialysis-desalted, and then lyophilized. This operation was repeated to secure an amount necessary for the test.
The non-adsorbed part is a neutral fraction (sample 1-N in Test Example 1), and the adsorbed part is an acidic fraction (sample 1-A in Test Example 1) that is an antiallergic agent of the present invention. did. The recovered amounts of the neutral fraction and the acidic fraction were approximately 1: 1, respectively. Further, the components of the acidic fraction and the composition of the constituent sugars were examined, and the obtained results are shown in Tables 1 and 2.

Uronic acid was converted to galacturonic acid by the m-hydroxydiphenyl method (Blumenkrantz et al.). Neutral sugars (rhamnose, fucose, arabinose, galactose, xylose, glucose, mannose) were quantified by GCMS as sugar alcohol acetate derivatives after TFA degradation. The galacturonic acid and neutral sugar measured by these methods were combined as the constituent sugar mass, and the ratio of each constituent sugar to this mass was taken as the constituent sugar composition.
The protein content was converted to BSA by Bradford, and the degree of esterification was measured by quantifying methanol after NaOH hydrolysis by GLC. The ash content was measured by ashing at 600 ° C. for 4 hours. Lipids were measured by the ethyl ether extraction method. The carbohydrate was 100- (water + protein + lipid + ash). As a result, most of the components of the acidic fraction were carbohydrates, the molecular weight cut off was as large as 6000 and adsorbed to the anion exchange resin, suggesting that the main component is acidic polysaccharides.

［試験例］

[Test example]

Sample analysis and anti-allergic activity assay The anti-allergic activity of Sample 1, Sample 1-N and Sample 1-A obtained in Example 1 was assayed by the PCA reaction inhibitory effect. Picryl chloride was used as a hapten (antigen lacking immunogenicity and only reactive), and the method of Lavaud et al. Was partially modified. According to this method, an allergic reaction can be observed without being affected by changes in immune indices such as IgE antibody production during the breeding period.
That is, a group of 6 7-week-old male BALB / c mice (Nippon Claire Co., Ltd.) preliminarily raised for 1 week, 0.1% of the standard powder feed CE-2 (Nippon Claire Co., Ltd.) or A feed mixed with various samples so as to be 0.2% (w / w) was freely ingested for 3 or 4 days, and then a PCA reaction inhibitory effect test was performed. A group fed only with the standard powdered feed CE-2 was used as a control.

  In addition to samples 1-N and 1-A, commercially available pectin and soybean polysaccharide were used for comparison. Polygalacturonic acid (Wako Pure Chemical Industries), LM12-1 (GENU pectin, Sankisha), HM (GENU pectin, Sankisha), lemon pectin (Wako Pure Chemical Industries), apple pectin (Wako Pure Chemical Industries) Yakuhin Kogyo Co., Ltd.) and soybean polysaccharide (SM-700, Saneigensha) were used.

The test of the PCA reaction inhibitory effect was performed as follows.
That is, 100 μL of 0.1% BSA (Nacalai Tesque) -containing phosphate buffer containing 2 μg of anti-TNP IgE (BD Pharmingen brand, Nippon Becton Dickinson) was injected into the mouse tail vein and left for 30 minutes. The thickness of the ear was measured using a thickness gauge (Ozaki Mfg. Co., Ltd.). Apply 20 μL of an acetone: olive oil mixture (1: 1 (v / v)) containing 0.8% picryl chloride to the ear of the mouse, leave it for 2 hours, and then measure the thickness of the ear again using a thickness gauge. (Ear thickness after reaction). The difference in ear thickness before and after the reaction was regarded as ear swelling, and the results of 6 animals in each group were statistically tested to evaluate the antiallergic effect. The results are shown in Table 3 as ++ when significant below and as-when there is no statistically significant difference.
The solubility in acid was determined by visual observation after dispersing 20 mg of a sample in a small amount of isopropanol, adding 1 ml of a pH 2 HCl solution, stirring in water at 25 ° C. for 5 minutes. The determination results are shown in Table 3 as + for those that were clearly dissolved, ± for a small amount of insoluble matter or cloudy, and − for those that clearly had turbidity or precipitation.

As shown in Table 3, the antiallergic activity was strongest in sample 1-A (acidic fraction), and the effect appeared 3 days after administration at a mixing ratio of 0.1% to the sample. On the other hand, sample 1-N (neutral fraction) had no antiallergic activity.
When sample 1 was administered at a sample contamination rate of 0.2%, anti-allergic activity was observed 4 days after administration, but no strong activity like sample 1-A (acidic fraction) was observed. It was. From the analysis results, it was understood that the xylose / galacturonic acid ratio was high, but it was a mixture of a neutral fraction containing xylose and no galacturonic acid, and an acidic fraction containing both xylose and galacturonic acid. .
The antiallergic activity of commercially available pectin and polygalacturonic acid used as comparisons was not observed even after 4 days of administration. The xylose content was low and the xylose / galacturonic acid ratio was 0.1 or less.
Further, soybean polysaccharide was dissolved in acid, but no antiallergic activity was observed. The xylose / galacturonic acid ratio was 0.14.
Therefore, 1) based on acidic polysaccharide, 2) soluble at pH 2, and 3) contains galacturonic acid and xylose as constituents, and the weight ratio of xylose / galacturonic acid is 0.2 or more. It was confirmed that Sample 1-A (acidic fraction) having the characteristics as described above was useful as the antiallergic agent of the present invention.

Manufacture of antiallergic agents (2)
After desalting soy sauce (Higashimaru Soy Sauce Co., Ltd.), the precipitate generated by adding 3 times the amount of ethanol was dried to obtain a soy sauce polymer material (Sample 2). This was dispersed in pH 2 water and separated into acid-soluble and acid-insoluble substances by centrifugation. Further, when an acid-soluble substance was fractionated on an anion exchange resin by a DEAE column in the same manner as in Example 1, 95% or more of the acid-soluble substance was obtained as an acidic fraction. The obtained acidic fraction was desalted and then dried to obtain an acidic fraction (sample 21A). Further, a part of the acidic fraction (sample 21-A) was fractionated by molecular weight using vivaspine (Sartorius). It was divided into three fractions of 10,000 or more, 1-0.5, and 50,000 or less.
For each of these samples, analysis and measurement of antiallergic activity were performed in the same manner as in Test Example 1, and the characteristics and antiallergic activity of each sample are shown in Table 4. All samples had a protein per dry matter of 1% or less, an ash content of 4-6%, a lipid of 0%, and a carbohydrate content of 93-95%.

As shown in Table 4, sample 21A (acid-soluble substance) was clearly more active than sample 2 (control). In addition, among the molecular weight fractions of Sample 21A, the activities of the fractions having a molecular weight of 10,000 or more and 1-50,000 were strong.
Since the fraction of sample 21A having a molecular weight of 50,000 or less had low activity, it was confirmed that it was necessary to contain a certain amount or more of galacturonic acid in order to exhibit strong antiallergic activity. The amount was 10% or more, preferably 15% or more, more preferably 20% or more, per weight of the constituent carbohydrate.

Manufacture of antiallergic agents (3)
After squeezing soy sauce moromi produced by regular methods using defatted soybeans and whole soybeans, and desalting raw soy sauce obtained by filtration, a double amount of ethanol was added to collect the generated precipitate. The precipitate was washed by adding 65% ethanol and then dried (Sample 3). This was dispersed in a pH 2 aqueous solution and then filtered, and a clear liquid was collected and dried. A part of this was fractionated in the same manner as in Example 1 to obtain an acidic fraction (Sample 3A). These samples were analyzed and the antiallergic activity was measured in the same manner as in Test Example 1. The constituent components of each sample are shown in Table 5, and the characteristics and antiallergic activity are shown in Table 6.

  As shown in Table 6, it was confirmed that the antiallergic activity of Sample 3A was clearly enhanced compared to the control product (Sample 3).

Results As shown in Examples 1 to 3 and Test Examples, the fractions in which strong antiallergic activity was observed in the present invention are all 1) mainly composed of acidic polysaccharides and 2) soluble at pH 2 3) It was a product containing galacturonic acid and xylose as components and having a xylose / galacturonic acid ratio of 0.2 or more.

  According to the present invention, 1) the main component is an acidic polysaccharide, 2) it is soluble at pH 2, and 3) it contains galacturonic acid and xylose as constituent components, and the weight ratio of xylose / galacturonic acid is 0.2 or more. By providing a novel antiallergic agent characterized in that, it is possible to provide a drug exhibiting stronger antiallergic activity than an antiallergic agent obtained from a conventional soybean fermented product such as soybean or soy sauce. .

Claims (3)

The antiallergic agent which has the characteristics of following 1) or 2) containing the acidic fraction obtained by the manufacturing method in any one of following (1)-(3) .
1 ) Soluble at pH 2
2 ) Containing galacturonic acid and xylose as constituent sugars of the acidic fraction, and the weight ratio of xylose / galacturonic acid is 0.2 or more
(1) A solution obtained by extracting soybean or soybean seed coat with acidic water is dialyzed (fraction molecular weight 6000), and an ethanol precipitate in the dialyzed internal solution is fractionated with an anion exchange resin to obtain an acidic fraction. .
(2) The acid-soluble fraction obtained by dispersing the ethanol precipitate of the soybean fermented product in acidic water is fractionated with an anion exchange resin to obtain an acidic fraction.
(3) Disperse ethanol precipitate of soybean fermented product in acidic water and filter the solution obtained by dialysis with a dialysis membrane with a molecular weight cut off of 6000 (fractionated molecular weight of 6000) and fractionating the acid fraction. Get a minute. The antiallergic agent according to claim 1, comprising 10% or more of galacturonic acid. The antiallergic agent according to claim 1 or 2, wherein the fermented soybean product is soy sauce.